Effects Of Signaling-selective Parathyroid Hormone Analogs Peptide On Fracture Healing In Orchiectomied Male Mice

BackgroundOsteoporosis, characterized by the loss of bone mass and bone degradation of microscopic structure which results in the increase of bone fragility and fracture,is a systemic skeletal disease. Osteoporosis is divided into two categories of primary osteoporosis and secondary osteoporosis. According to epidemiological surveys estimated that there are about75million people were suffering from osteoporosis now in the United States and Europe and Japan,the number of patients continues to increase in the future. The survey of National Osteoporosis Foundation (NOF) found that according to the WHO (World Health Organization) diagnostic criteria,there were54%post-menopausal white women were suffering from reduced bone mass and30%post-menopausal white women suffering from osteoporosis, more than3%to6%of the50-year-old male were suffering from osteoporosis. Prediction of WHO,over the age of65will reach900million in Asia in2050,3.2million patients will suffer hip fracture every year, which was mainly due to the loss of of bone mass and osteoporosis. With the aging of our population, osteoporosis has become one of the most serious public health problems. According to the result of supported by the National Key Technologies Research and Development program of China during the 9th Five-Year Period, Chinese prevalence of osteoporosis was22.6%, the loss of bone mass was13.3%. The serious consequences of osteoporosis are fractures and complications of fracture due to disability and death, which bring more painful to the patient, and bring a heavy burden to the family and society at the same time.The main treatment of osteoporosis which including basis treatment and medication.The basic treatment, including dietary adjustments,calcium and vitamin D, and so on. The basic principle of drug therapy is promote bone formation and inhibit bone resorption. Anti-resorptive drugs used in clinical such as estrogen, bisphosphonates, calcitonin, activated vitamin D derivatives and so on. Promoting bone formation drugs, such as fluorides, male hormone and its derivatives, growth hormone, parathyroid hormone. In the metabolism of drugs of promote bone formation and increase bone mass, PTH is the drug which was unanimously approved,(although the strontium salt is considered to have a certain role in promoting bone formation, but more research is needed to confirm).PTH is an essential regulator of calcium homeostasis. PTH(1-34), the truncated form of nature PTH(1-84), with the same binding ability and same potential to activate type I PTH receptor (PTHR1), has been identified as a powerful anti-osteoporosis agent. PTH (1-34) has been put into clinical in the treatment of osteoporosis in the United States, and formally use in our country in October2011. Clinical follow-up observation found that it has reliable anti-osteoporosis effect, but has not yet reached the desired effect. One of the important reasons is the role of PTH on bone tissue is bidirectional, that intermittent small doses promote bone formation and continuous high-dose stimulates will promote bone resorption. PTH regulate osteoblast generation, conversion, proliferation or apoptosis, also promote osteoclast resorption. The main function of PTH on bone tissue in combination with type1PTH receptor (PTHR1) which exist in the surface of osteoblasts, and the role of osteoclasts indirectly through osteoblasts. PTH affect bone formation and resorption was interrelated with PTHR1cause multiple signaling pathways. After binding with PTHR1, several G protein coupled signaling cascades including cAMP/PKA, PLC/PKC and PLC-independent/PKC are triggered by PTH. Each pathway has been mapped to different key domains within the PTH(1-34) aminoacid sequence. Serine in position1is crucial in PLC activation, and the first3amino acids at the N terminus are required for cAMP/PKA activation. Our previous study identified that PTH(29-34) is important for PLC-independent/PKC signaling. With the understanding of the signaling selective properties in the domains of PTH(1-34),[Glyl,Arg19] hPTH (1-34), an analog lacking the ability to activate the PLC signaling pathway. Serine1(Ser1) mutation to glycine (Gly1) is significantly lose the characteristics of activation of PLC signal.Glutamate19acid mutation into arginine (Arg19) can be compensated the weakening of the receptor binding of29-34amino acid residues missing. This conclusion has been confirmed by early experients, intermittent small dose injection of PTH and its alterations defective analog peptide increase the number and size of the trabecular bone below the tibial plateau and increased body bone mineral density.There are many animal studies reported that PTH(1-34) to promote healing of fracture, such as intermittent injection of PTH (1-34) improve bone mineral density and bone callus formation, promote fracture healing and increased body bone mineral density, affect serum osteogenic formation marker. Not yet study reporte PTH analog peptide role in fracture healing.Androgen has a wide range of physiological effects in vivo, especially in male genital development, sexual maturation and spermatogenesis maintain. The main component of the androgen is testosterone. Studies reported that testosterone has an important influence for human bone growth and development,but with age increased, the concentration of testosterone in the human body is gradually reduced, the older men of the low levels of testosterone concentrations easily lead to accelerate bone turnover and increase the risk of fractures. The research about the relation of low testosterone levels bone micro-structural changes and fracture healing is very limited. The use of the signal selectivity parathyroid hormone intervention in animals after fracture and observation of fracture healing will help us conducive to the understanding of the signaling pathway for fracture healing, explore the design and use of new drugs. Objectives1. To establish Orchiectomized model in male mice,and anysis the change of serum testosterone level and BMD and micro-strcture between orchiectomized group and sham group.2. In this study, a PLC deficient PTH analog,[GlylArgl9] hPTH(1-34)(G1,R19(1-34)) was synthesized and its effect on bone fracture healing in the femoral shaft of orchiectomized male mice was investigated and compared with hPTH(1-34), to see the role of PLC in PTH’s effects on bone healing and to promote the development of drugs research and to provide new types of treating fracture.Methods1. Orchiectomized male mice model and femur fracture model407-week-old C57BL/6J male mice, eight of them as the baseline group, the remaining thirty-three were randomly divided into orchiectomized group and sham group, the experiment is agreed by the NanFang Hospital ethics committee of animal experiments. Mice were anesthetized with an animal anesthesia machine by Flurane inhalation.Routine skin preparation, skin disinfection, cut about1cm in the skin of the scrotum midline, exposure bilateral testes, epididymis and surrounding tissue, and removal of the testicles,then sutured the scrotum surgery skin disinfection. Sham group, only cut the skin of the scrotum stitched:Established the middle femur fracture model after castration one week later, isoflurane gas anesthetized, and choose the skin of right thigh preparation, skin disinfection, according to the mouse femur fracture model reported in the literature, a cross-sectional fracture was generated in the mid-shaft of the right femur.Timing observed after wound healing, all the mice were not infection, incision healed well. Two weeks and four weeks after the fracture, the removal of the mouse eyes and blood serum was separated.The concentration of serum androgen (testosterone) was measured by ELISA test, using Micro-CT to measure of bone mass and bone microarchitecture under tibial plateau in2mm and fracture healing.Application SPSS13.0statistical software for data processing, the results are presented as mean±SEM, groups were compared using two independent samples t test was used for statistical analysis.The difference was statistically significant at P<0.05.2. Effects of signaling-selective parathyroid hormone analogs peptide on fracture healing in orchiectomied male mice487-week-old C57BL/6J male mice were orchiectomized. The bone fractures were made in mid-shaft femur then a small rod inserted in to the bone marrow cavity to stabilize the fracture. The once daily injections of hPTH (1-34)(40ug/kg), G1,R19(1-34)(40ug/kg) or vehicle (0.05ml) started at the second day and lasted for4weeks (5times/week). The bone mineral content (BMC) and density (BMD) in fracture healing area were measured with dual energy X ray densitometer and micro CT. The bone healings at week2or4were demonstrated by radiography, biomechanics testing, micro-CT, and histology.Resuts1. One week after orchiectomy, the male mice exhibit slow movement and eagerness to huddle together, but they maintain a similar food and water intake to the sham-operated mice. Two weeks after fracture, the serum testosterone level (186.16±6.47ng/L) was significantly reduced compared with that of the sham animals (215.83±8.36ng/L). Four weeks after frature, serum testosterone level decreased further (94.34±3.96ng/L).In order to assess the bone metabolism associated with the testosterone loss, the trabecular bone2mm below the growth plate of the tibia was investigated. Less trabecular bone and thinner cortical bone were seen in orchiectomized mice. Bone volume (BV/TV) dramatically decreased in orchectomized mice but increased in sham from week2to week4. At week2, the trabeculae2mm below the growth plate of tibia in orchiectomized mice were less than in the sham mice. The BV/TVs in bone healing area were significant lower in orchiectomized mice (p<0.05). At week4, the trabeculae continued decreasing and the difference was much clearer (p<0.05). In the fracture area, BV/TVs increased both in orchiectomized and sham mice, while latter have more BV/TV (p<0.01).2. X ray confirmed all the fractures were lined up well in all the animals. At week2, BMD (45.92±1.62) and BMC (10.33±0.61) in PTH(1-34) treatment group were significantly (p<0.05) increased as compare with the vehicle treated group (BMD33.77±2.17/BMC8.48±0.17), but less than that with G1,R19(1-34) treatment (BMD49.12±2.11/BMC13.51±1.05, p<0.05). CT scanning showed that more cancellous bones in hPTH(1-34) and G1,R19(1-34) treated groups at week2consistent with BMD and BV/TV detected with micro CT. And at this time, G1,R19(1-34) has significant higher value in BMD (p<0.05). At week4, the healing bone almost finished reconstruction in hPTH(1-34) and G1,R19(1-34) treated groups with higher BMD and BV/TV, but no difference remained between hPTH(1-34) and G1,R19(1-34). In HE staining, more trabeculae in hPTH(1-34) and G1,R19(1-34) treatment groups and at week4,bone marrow cavity rechanneled in hPTH(1-34) and G1,R19(1-34) groups but still under reconstruction in vehicle group.ConclusionsOrchiectomy, with androgen deficiency, induces bone loss and retarded bone fracture healing in male mice. G1r19(1-34) could not activate PLC signaling, but have the more than equivalent effect on fracture healing as PTH(1-34), which means that PLC signaling pathway is not required in PTH’s effect on bone healing...